Publications and other reference materials referred to herein are incorporated herein by reference and are numerically referenced in the following text and respectively grouped in the appended Bibliography which immediately precedes the claims.
The detection of small quantities of a substance in solution can be accomplished by fluorescence labeling. For example, the detection of analytes in human serum has been achieved by time-resolved fluoroimmunoassay or fluorescence polarization immunoassay (Ref. 1 to 3).
Dyes which have been used extensively as fluorescent labels in probes and immunoassays include fluorescein derivatives such as fluorescein isothiocyanate, rhodamine derivatives and derivatives of the chelates of rare earth metals such as europium. (Ref. 5, 7)
Certain physical and chemical properties of a dye may contribute to determining its overall utility as a fluorescent label, e.g. for homogeneous fluoroimmunoassay and/or probe in the detection of analyte. Important properties include fluorescence intensity, fluorescence lifetime, excitation and emission wavelength maxima, polarization and non-specific binding behavior.
(a) Fluorescence Intensity: The intensity of the fluorescence produced upon excitation of the probe with light (such as from a laser source). The nature of the solvent used for the fluorescence measurement may have a significant effect on the intensity of the fluorescence of a given probe. Use of aqueous solvent systems, such as biological buffers is convenient and possibly essential in applications such as immunoassays. Self-aggregation of the probes in these solvents may substantially attenuate their fluorescence intensity.
(b) Excitation and Emission Wavelengths: The wavelength of light required to efficiently produce fluorescence and the wavelength of light at which fluorescence emission occurs. (Fluorescence emission occurs at a longer wavelength than the excitation wavelength.) Ultraviolet, visible and infrared light (typically wavelengths in the range of about 200 nanometers to about 1000 nanometers) are considered to be wavelengths which are potentially useful in exciting a dye molecule and thereby producing detectable fluorescence. Due to the abundance of naturally occurring substances which fluoresce upon excitation at relatively short wavelengths (in the range of about 200 nm to about 500 nm), improved sensitivity of detection may be achieved by using a probe having a fluorophore which fluoresces upon excitation by light of wavelength greater than about 500 nm, preferably in the spectral range of about 500 nm to about 900 nm. (Ref. 5)
(c) Fluorescence Lifetime and Fluorescence Decay Time: The lifetime of the fluorescence produced by the probe may vary, from less than one nanosecond to several milliseconds. Most organic dyes which exhibit fluorescence lifetimes in the range of 3 to 50 nanoseconds belong to the general class of compounds commonly referred to as aromatic compounds and are exemplified by aromatic hydrocarbon derivatives such as perlene carboxylic acid and aromatic heterocyclic compounds such as phthalocyanines and naturally occurring porphyrins. These dyes have a characteristic fluorescence lifetime, that is, the time period following excitation during which they emit light and during which the fluorescence intensity decreases to about 37% (l/e) of its initial value in the absence of any deactivating factors. The measured fluorescence decay time is the time period during which the decrease to the 37% (l/e) level of fluorescence intensity is observed in realistic situations. The measured decay time of a particular compound may be solvent dependent. Under conditions which minimize deactivation, measured decay time may approach fluorescence lifetime. In order to be suitable for use in an assay such as a fluorescence polarization immunoassay, the measured fluorescence decay time (and necessarily fluorescence lifetime also) of the probe must be suitably long (at least about 2 nanoseconds, preferably on the order of about 20 nanoseconds). Additionally, probes having extended fluorescence decay times allow for improved detection of signal relative to the natural fluorescence background of a sample containing serum.
(d) Fluorescence Polarization: When a fluorescent substance in solution is excited with polarized light, it emits partially polarized light as fluorescence. The degree of polarization of fluorescence can be measured, and is related to the molecular volume of the fluorophore. This relationship can be used to determine the extent of binding of small fluorescent probes serving as haptens to relatively large antibodies.
(e) Non-Specific Binding: The ability of the probe to remain unbound in solution in the presence of large protein molecules such as human serum albumin (MW about 70,000). Nonspecific binding of the small (low molecular weight) dye molecules to relatively large (high molecular weight) macromolecules such as proteins in solution has been observed. (Ref. 14) The occurrence of this noncovalent association between a fluorescent probe and a biological macromolecule is manifested in the fluorescence behavior of the probe-macromolecule complex. Generally, fluorescence polarization is affected. In applications such as homogenous fluorescence immunoassays, it is essential that non-specific binding of the probe to biological macromolecules is kept to a minimum, if not eliminated.
Non-specific binding in immunoassays has always been a troublesome problem, especially if serum samples are involved. In order to circumvent the difficulties caused by non-specific binding, investigators in the past have resorted to the addition of various surfactants such as sodium dodecyl sulfate and chaotropic ions such as potassium trichloracetate or to the precipitation of the serum proteins with protein precipitants such as sulfosalicylic acid followed by a separation step such as centrifugation or filtration to remove the precipitate.
While a separation step solves the problem of nonspecific binding it makes the assay time-consuming, expensive and difficult to automate. Also, the various additives mentioned invariably interfere not only in the non-specific binding but to varying extents in the specific binding by antibody as well. this effect is fully to be expected since the types of interactions in specific and non-specific binding are the same, viz., electrostatic, hydrogen bonding and hydrophobic interactions. Hence, these methods have not proven satisfactory, some differential effect being the best that can be expected.
Most of the non-specific binding of serum is due to the presence of serum albumin. This protein has several specialized functions among which is the carriage of a variety of organic metabolites such as fatty acids and steroid hormones and in addition it carries other substances ingested orally or received by injection. The structure of serum albumin is somehow uniquely suited to this function and when tested in vitro can be shown to bind nearly every type of molecule up to a molecular weight range of a few thousand.
Organic dyes, especially those with aromatic and/or polycyclic structures, may have limited solubility in aqueous solutions and may aggregate in aqueous solvents. (Ref. 4) Fluorescent dyes which are hydrophobic can be modified in order to promote solubility in water; for example, by sulfonation of the dye or dye precursors. (Ref. 6) In general, where an organic molecule has limited solubility in water, improved solubility can be achieved by chemically bonding the organic molecule to one or more water solubilizing groups. Additional examples of such groups are phosphate, carboxylate and quaternary ammonium, and their salts such as sodium and potassium phosphates and ammonium halides.
Porphyrin and phthalocyanine derivatives are useful as fluorophores and exhibit good fluorescent behavior in organic solvents. However, underivatized porphyrin and phthalocyanine compounds are essentially water insoluble and, thus, are highly aggregated in aqueous solutions and exhibit low fluorescence intensity. Modification of porphyrin and phthalocyanine compounds by attaching solubilizing groups such as sulfonate, quaternary ammonium, carboxylate and the like, has improved the water solubility of these compounds; however, the derivatized porphyrins and phthalocyanines often exhibit substantially decreased fluorescence intensity in aqueous solution as opposed to in organic solvents. In addition the water-soluble derivatives have been found to bind tightly in a non-specific manner to components of human serum which limits their usefulness in immunoassays.
Phthalocyanine and porphyrin derivatives have found application in the area of tumor therapy (Ref. 8,9). Water soluble derivatives of these compounds have been prepared. For example, the metallated and sulfonated phthalocyanine, hydroxyaluminum phthalocyanine tetrasulfonic acid, is essentially monomeric in aqueous solutions and efficiently produces fluorescence in water. (Ref. 10, 16). Without exception, however, these previously described fluorophores retain a strong tendency to bind nonspecifically to solution components such as human serum albumin.